U.S. patent number 4,987,237 [Application Number 07/112,742] was granted by the patent office on 1991-01-22 for derivatives of monophosphoryl lipid a.
This patent grant is currently assigned to Ribi ImmunoChem Research, Inc.. Invention is credited to Kent R. Myers, Edgar F. Ribi, deceased.
United States Patent |
4,987,237 |
Myers , et al. |
January 22, 1991 |
**Please see images for:
( Certificate of Correction ) ** |
Derivatives of monophosphoryl lipid A
Abstract
Novel derivatives of monophosphoryl lipid A and a process for
their preparation are provided. The derivatives contain one or more
free groups, such as an amine, on a side chain attached to the
primary hydroxyl groups of the monophosphoryl lipid A nucleus
through an ester group. The derivatives provide a convenient method
for coupling the lipid A through coupling agents to various
biologically active materials, substrates, and the like, wherein
the immunostimulant properties of lipid A are desired.
Inventors: |
Myers; Kent R. (Hamilton,
MT), Ribi, deceased; Edgar F. (late of Hamilton, MT) |
Assignee: |
Ribi ImmunoChem Research, Inc.
(Hamilton, MT)
|
Family
ID: |
22345625 |
Appl.
No.: |
07/112,742 |
Filed: |
October 22, 1987 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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732889 |
May 8, 1985 |
4866034 |
|
|
|
526967 |
Aug 25, 1983 |
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Current U.S.
Class: |
549/222; 530/300;
530/322; 530/331; 536/117; 536/17.7; 536/18.7; 546/22; 548/119;
548/413; 548/414 |
Current CPC
Class: |
A61K
45/05 (20130101); C07H 13/06 (20130101); C07K
9/001 (20130101); C07K 17/06 (20130101) |
Current International
Class: |
A61K
35/66 (20060101); A61K 35/74 (20060101); A61K
45/00 (20060101); C07K 9/00 (20060101); C07H
13/06 (20060101); C07H 13/00 (20060101); C07K
17/00 (20060101); C07K 17/06 (20060101); A61K
38/00 (20060101); C07F 009/655 (); C07F
009/6558 () |
Field of
Search: |
;549/222
;548/119,413,414 ;546/22 ;530/331,300,322 ;536/17.7,18.7,117 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
"Peptides as Requirements for Immunotherapy . . ." E. Ribi et al.,
Cancer Immunology & Immunotherapy, 1979--pp. 43-58. .
"Heterogeneity & Biological Activity . . ." C. Chen et al.,
Journal of Infect. Diseases, 1973--pp. S43-S51. .
"Beneficial Modification . . ." E. Ribi, Jour. Bio. Rspn., pp. 1-9.
.
"Chem. Comb. of Biol. Active Der . . .", Hasegawa et al., 1986, pp.
371-385..
|
Primary Examiner: Raymond; Richard L.
Attorney, Agent or Firm: Burgess, Ryan & Wayne
Parent Case Text
RELATED APPLICATIONS
This application is a continuation-in-part of application Ser. No.
732,889 filed May 8, 1985, now U.S. Pat. No. 4,866,034, which is a
continuation-in-part of application Ser. No. 526,967 filed Aug. 25,
1983, now abandoned. Both applications are incorporated herein by
reference.
Claims
What is claimed is:
1. A derivative of monophosphoryl lipid A having the formula:
##STR16## wherein R.sup.1 and R.sup.2 are hydrogen, R.sup.3 is a
straight or branched hydrocarbon chain composed of carbon,
hydrogen, and optionally oxygen, nitrogen and sulfur which if more
than one atom may be the same or different,
wherein the total number of carbon atoms does not exceed 60, and
the circle represents a monophosphoryl lipid A nucleus.
2. A derivative of monophosphoryl lipid A having the formula:
##STR17## wherein the segment of the derivative represented by:
##STR18## contains 2-60 carbon atoms and wherein R.sup.3 is a
straight or branched hydrocarbon chain composed of carbon,
hydrogen, and optionally oxygen, nitrogen and sulfur which if more
than one atom may be the same or different, and x is a minimum of 1
and can be any whole number such that the total number of carbon
atoms of all x segments does not exceed 60, wherein the chemical
structure of each R.sup.3 may be the same or different in each such
segment and wherein the circle represents a monophosphoryl lipid A
nucleus.
3. The derivative of claim 1, wherein the total number of carbon
atoms in R.sup.3 does not exceed 20.
4. The derivative of claim 2, wherein the segment of the derivative
represented by: ##STR19## contains 2-20 carbon atoms and wherein
R.sup.3 is as indicated and wherein x is a minimum of 1 and can be
any whole number such that the total number of carbon atoms of all
x segments does not exceed 20, and wherein the chemical structure
of each R.sup.3 may be the same or different in each such
segment.
5. The derivative of claim 2, wherein the segment of the derivative
represented by: ##STR20## is an alpha-amino acid residue and
wherein when the value of x is greater than 1, the alpha amino acid
residue in each such segment may be the same or different.
6. The derivative of claim 4, wherein the segment of the derivative
represented by: ##STR21## is an alpha-amino acid residue and
wherein when the value of x is greater than 1, the alpha-amino acid
residue in each such segment may be the same or different.
7. The derivative of claim 3, having the formula: ##STR22## wherein
x is 5 and the circle represents a monophosphoryl lipid A
nucleus.
8. The derivative of claim 5, wherein the alpha-amino acid residue
is selected from the group consisting of glycine, alanine,
methionine, valine, norvaline, leucine, isoleucine, phenylalanine
and lysine.
9. The derivative of claim 6, wherein the alpha-amino acid residue
is selected from the group consisting of glycine, alanine,
methionine, valine, norvaline, leucine, isoleucine, phenylalanine
and lysine.
10. The derivative of claim 9, wherein the alpha-amino acid residue
is glycine.
11. The derivative of claim 9, wherein the alpha-amino acid residue
is lysine.
Description
FIELD OF THE INVENTION
This invention relates in general to certain novel derivatives of
monophosphoryl lipid A. In one aspect, this invention is directed
to novel derivatives of monophosphoryl lipid A containing one or
more substituted or unsubstituted amino groups. In another aspect,
the invention relates to novel derivatives which are conjugates of
monophosphoryl lipid A with certain biologically active materials.
In a further aspect, this invention relates to methods for the
preparation and use of the derivatives of this invention.
BACKGROUND OF THE INVENTION
Prior to the present invention, numerous reports have appeared in
the literature which cited the reaction of lipopolysaccharide (LPS)
with cyclic anhydrides, especially phthalic and succinic anhydride.
However, with the exception of one reference, no mention has been
made of combining monophosphoryl lipid A (MPL) with these or other
anhydrides.
In an article by A. Hasegawa, et al, J. Carbohydrate Chem.5: 371,
there is disclosed the covalent coupling of muramyl dipeptide (MDP)
to a derivative of lipid A corresponding to the non-reducing
glucosamine residue. This lipid A derivative, designated GLA-27 in
the reference, was blocked at both the phosphate and at all other
potentially reactive positions except for the hydroxyl to which
coupling was desired (i.e. the primary hydroxyl at C6). The
coupling strategy employed in this reference involved introducing a
free carboxyl group into GLA-27 at C6 via succinic anhydride and a
free amine into MDP, and then forming an amide linkage between
these two groups. A key difference between the teachings of this
reference and those of the present invention are that in the
reference, the lipid A derivative had to be completely blocked in
order to avoid side reactions during the condensation (i.e., amide
bond forming) step. The present invention avoids this difficulty by
introducing a free amino group into MPL. Coupling of MPL to other
components is then achieved using reagents which react specifically
with amines (e.g. aldehydes), or else activating an appropriate
group on the other component prior to combining with the
amino-MPL.
Acylation of lipopolysaccharides by cyclic anhydrides probably
occurs in the O-antigen and core regions which are absent in
monophosphoryl lipid A. The purpose of acylating LPS in these prior
art references was to attenuate its toxicity, not to introduce a
functional group for the purpose of forming covalent conjugates
with other materials.
Accordingly, one or more of the following objects will be achieved
by the practice of the present invention. It is an object of the
invention to provide certain novel derivatives of monophosphoryl
lipid A and a process for their preparation. Another object of this
invention is to provide a novel process for the preparation of
derivatives of monophosphoryl lipid A which does not require the
blocking of any functional groups in order to introduce the desired
groups into the molecule.
A still further object of the invention is to provide novel
derivatives which are conjugates of monophosphoryl lipid A with
biologically active materials such as antigens, antibodies,
immunomodulators and the like. Another object is to provide novel
conjugates of the derivatives with biological compounds such as
antigens, which exhibit enhanced biological activity.
A further object of this invention is to provide a process for the
preparation of derivatives of monophosphoryl lipid A in relatively
pure form and substantially free of undesirable reaction
by-products.
Another object of the invention is to provide methods for using the
conjugates. These and other objects will readily become apparent to
those skilled in the art in light of the teachings contained
herein.
SUMMARY OF THE INVENTION
In its broad aspect, the invention relates to certain novel
derivatives of monophosphoryl lipid A, certain intermediate
compounds, a process for their preparation and use thereof. The
novel derivatives are represented by the formula: ##STR1## wherein,
Z and R and the circle are as hereinafter defined.
These derivatives are conventionally prepared by reacting
monophosphoryl lipid A with a compound containing a group or groups
which is (are) predisposed to undergo a bond-forming reaction with
a group or groups present in MPL. The use of a compound which is
activated prior to the reaction with MPL avoids the necessity of
first introducing protecting groups into the MPL molecule.
DETAILED DESCRIPTION OF THE INVENTION
Monophosphoryl lipid A (MPL) is a phosphorus-containing
polyheterocyclic compound having pendant long chain, aliphatic
ester and amide groups, and is obtained as an endotoxic extract
from Enterobacteriaciae. The compound, also referred to as "MPL" is
prepared in a manner set forth in U.S. Pat. Nos. 4,436,727 and
4,436,728 which are incorporated herein by reference. Endotoxin
extracts of the type used as the starting material to produce MPL
may be obtained from any Enterobacteriaciae including parent
organisms and mutants. The aforesaid patents describe the type of
microorganisms that may be used to obtain the starting material and
several methods for preparing the starting material. MPL may also
be prepared by synthetic and genetic engineering techniques. The
preferred method to date of obtaining the endotoxic extract is that
disclosed by Chen, et al, J. Infect. Dis. 128 543 (1973).
MPL is a composition characterized as having no detectable
2-keto-3-deoxyoctanoate, between about 350 and 475 nmoles/mg of of
phosphorus and between about 1700 and 2000 nmoles/mg of fatty
acids. Although the process for the preparation of refined
detoxified endotoxin (MPL) was disclosed and claimed in the
above-mentioned U.S. Pat. No. 4,436,727, the chemical structure was
not fully known at that time and accordingly, it was necessary to
describe MPL as a product-by-process. The complete structure of the
hexaacyl form of MPL obtained from lipopolysaccharides of
Salmonella minnesota R595 is now known and has been given as
follows: ##STR2##
MPL is a significant improvement over endotoxic extracts from
Enterobacteriaciae because MPL is detoxified and therefore does not
contain the highly toxic components which have rendered endotoxic
extracts unsuitable for therapeutic use. (See Peptides as
Requirements for Immunotherapy of Guinea Pig Line-10 Tumor with
Endotoxins, Ribi, et al, Cancer Immunol. Immunother., Vol. 7, pp.
43-58, 1979, incorporated herein by reference.) The beneficial
effects of MPL over other endotoxic extracts are described for
example in U.S. Pat. Nos. 4,436,727 and 4,436,728 and in Ribi, E.,
Journal of Biological Response Modifiers, Vol. 3, pp. 1-9, 1984
(incorporated herein by reference). Due to the fact that the
monophosphoryl lipid A is non-toxic, it has recently found
application as an adjuvant for vaccines, and in other
pharmaceutical compositions useful in the treatment of various
disorders such as cancerous tumors in warm-blooded animals and the
like.
The effectiveness of MPL as an adjuvant for certain antigens may be
enhanced by coupling MPL directly to these antigens. In addition,
covalent conjugates consisting of MPL and other immunopotentiating
compounds may exhibit biological activities which are enhanced
relative to the free (non-conjugated) components.
Traditional methods of forming covalent conjugates can be divided
into two categories depending on whether the molecules are
activated with respect to coupling prior to being mixed or are
activated in situ after being mixed. Neither approach is feasible
with unmodified MPL because of its structural configuration. For
example, the prior activation approach entails introducing a group
into one of the molecules which will react readily with a group
present only in the second molecule such as an amino or a
sulfhydryl. This approach is not feasible with unmodified MPL since
it does not contain any functional groups which can be activated or
which will react with groups previously activated on other
molecules. The second approach also does not work with MPL since
under the conditions generally employed for in situ activation, MPL
tends to react with itself, which leads to a number of undesirable
side products and greatly reduces or altogether eliminates the
yield of the desired conjugate.
However, an appropriate functional group such as a primary amine
can be incorporated into MPL by the method of the present
invention; this allows normal coupling procedures to be employed
for purposes of attaching other materials to the resulting modified
MPL.
The functional groups which MPL contains include a primary hydroxyl
at C-6', between 3 and 6 secondary hydroxyls and one phosphate
group at C-4'. Due to the complex configuration of the
monophosphoryl lipid A molecule and in order to simplify the
reactions which occur in the process of the present invention, the
monophosphoryl lipid A molecule will be depicted as: ##STR3## with
the single primary hydroxyl shown at the top, a representative
secondary hydroxyl at the bottom, the phosphate group shown by
OPO.sub.3 H.sub.2, and the circle representing the remainder of the
monophosphoryl lipid A molecule.
As hereinbefore indicated, the derivatives of the present invention
are conveniently represented by the formula: ##STR4## wherein Z
represents hydrogen or the group:
wherein R represents a divalent group comprised of carbon, hydrogen
and optionally, one or more of oxygen, nitrogen or sulfur and
contains from 2 to 60 carbon atoms, preferably 2 to 20 carbon
atoms; A represents a divalent coupling group which is capable of
coupling R to Y through at least two independent functional groups,
and wherein A contains from 2 to 60 carbon atoms and optionally,
one or more of oxygen, nitrogen or sulfur; Y represents a
biologically active material, such as antigens, antibodies,
immunomodulators and the like; and n is zero or 1.
One embodiment of the present invention is directed to derivatives
within the scope of the above general formula when Z is hydrogen.
These monophosphoryl lipid A derivatives contain an ester side
chain attached to the carbon atom of lipid A which bore the primary
hydroxyl group (i.e., the C-6' carbon atom). The compounds can be
represented by the formula: ##STR5## wherein R represents a group
comprised of carbon, hydrogen and optionally, oxygen, nitrogen
and/or sulfur, and contains from 2 to 60 carbon atoms, preferably 2
to 20 carbon atoms.
In general, any type of functional group can be introduced into MPL
or related materials by the method of the present invention,
provided that the functional group is part of a molecule that
contains a carboxyl or similar group that can be activated toward
ester bond formation prior to combination with MPL. Functional
groups which can be introduced by this method include, but are not
limited to, amines, thiols, aldehydes, carboxyls, N-hydroxyimido
esters, imino esters, aryl azides, maleimides, pyridyl disulfides
and active halogens.
This requirement of activation, prior to combining with MPL, is of
key importance due to the numerous side reactions which can occur
if MPL is exposed directly to activating conditions. An example of
this prior activation approach is the reaction of MPL with succinic
anhydride, which results in the introduction of a free carboxyl
group into MPL. ##STR6## Thus, derivatives such as those of Formula
IV wherein R of Formula III terminates in a free carboxyl group and
is attached through the ester group to the MPL molecule by means of
an aliphatic, aromatic or heterocyclic group can be prepared in
accordance with the teachings of the present invention.
It may or may not be necessary to block certain functional groups
in the molecule which is to be introduced into MPL prior to the
activation step. For example, in introducing an amino group into
MPL by the method of the present invention, it is necessary to
block the amine of an appropriate amino acid prior to activating
the carboxyl for ester bond formation. In the example given above
involving succinic anhydride, both the blocking and prior
activation requirements are satisfied by formation of the cyclic
anhydride. Suitable blocking and deblocking procedures are well
known to those skilled in the art. See, for example, Theodora W.
Green, "Protective Groups in Organic Synthesis", John Wiley &
Sons, New York, 1981, pp. 349.
Preferred compounds which can be prepared by the process of the
present invention are the amines and substituted amines which can
be exemplified by the formula: ##STR7## wherein R.sup.3 represents
a divalent chain which contains carbon, hydrogen and optionally
oxygen, nitrogen and/or sulfur; and R.sup.1 and R.sup.2
individually represent hydrogen, or lower alkyl, or one of R.sup.1
or R.sup.2 can represent the residue of an organic substrate or
carrier including liposomes, or a biological material such as an
antigen, immunomodulator, antibody and the like.
Particularly refined compounds of Formula (V) are those wherein
R.sup.1 and R.sup.2 are hydrogen and R.sup.3 is a divalent radical
of from 2 to 60, and more preferably 2 to 20 carbon atoms and is
composed of carbon, hydrogen and optionally oxygen, nitrogen and/or
sulfur. These derivatives include those wherein R.sup.3 is a
straight or branched hydrocarbon chain, and polypeptides containing
one or more recurring amide groups, such as di- and tri-peptides of
glycine derivatives of MPL. Included within these are those
compounds wherein an amino acid is introduced into MPL at the C-6'
position and accordingly, provide MPL derivatives having a side
chain terminating in a primary amine. Typical compounds of this
type include: ##STR8## wherein x can be equal to any value from 1
up to about 20, and higher. One example of this compound is when x
=5, which corresponds to the ester of 6-amino caproic acid. This
compound is referred to as CAP-MPL and the synthesis is set forth
in Examples 1-3.
Another compound of interest is the derivative where MPL is
esterified to the free carboxyl end of peptides consisting of
glycine or related alpha-amino acids. For example, the following
compound consists of MPL esterified to a tripeptide of glycine:
##STR9##
Accordingly, a variety of derivatives of MPL can be prepared from
known amino acids. Alternative amino acids which can be introduced
into the MPL molecule by the process of the present invention
include, among others, glycine. alanine, serine, threonine,
methionine, valine, norvaline, leucine, isoleucine, phenylalanine,
tyrosine, crysteine, aspartic acid, glutamic acid, hydroxyglutamic
acid, arginine, lysine, cystine, histidine, proline,
hydroxyproline, tryptophan, asparagine, and glutamine. Suitable
blocking and deblocking procedures well known to those skilled in
the art may need to be employed when attaching these amino acids,
or combinations thereof, to MPL by the process of the present
invention.
Derivatives of MPL in which a primary amino group is introduced by
the method of this invention can be further modified to a form
which can be conveniently coupled to other compounds of interest,
such as antigens, antibodies and immunomodulators. The general form
of such MPL derivatives is as follows: ##STR10## wherein R.sup.1
and R.sup.3 are as hereinbefore described; R.sup.2 represents a
group comprised of carbon, hydrogen and optionally oxygen, nitrogen
and/or sulfur, and contains from 1 to 60 carbon atoms, preferably 1
to 20 carbon atoms; and R.sup.4 represents a group which is able to
form a covalent bond, either selectively with amines or sulfydryls
or non-selectively with a wide variety of groups, and which is
comprised optionally of carbon, hydrogen, nitrogen, oxygen and/or
sulfur. The radical denoted by R.sup.4 -R.sup.2 represent, for
example, N-succinimidyl suberoyl, 6-(4'-azido-2'-nitrophenyl)
hexanoyl, 3-(2-pyridyldithio propionoyl and m-maleimidobenzoyl.
Example 4 describes the preparation of the following compound:
##STR11## wherein R.sub.4 -R.sub.2 represents N-succinimidyl
suberoyl; and R.sub.1 and R.sub.3 correspond to the designation
given in Formula VIII with X =5.
Thereafter, the derivative comprised of MPL with a coupling group
attached to MPL through an amine group, such as SSC-MPL above, is
further reacted with a peptide containing a primary amine group to
form a conjugate: ##STR12##
The process for preparing the present invention, as hereinafter
described, provides a unique method for introducing one or more new
functional groups into monophosphoryl lipid A and related
compounds. These functional groups can provide a point of
attachment for covalent coupling of MPL and related compounds to
other materials of interest.
The process comprises the steps of:
(1) independently blocking the other functional group(s) of an
appropriate carboxylic acid, if necessary;
(2) activating the free carboxyl of the blocked compound with
respect to ester bond formation;
(3) reacting the activated acid with monophosphoryl lipid A;
and
(4) de-blocking the resulting MPL derivative, if necessary.
The process of the invention can be illustrated by the following
sequence of reactions wherein an amino-MPL derivative of the type
represented by Formula IV is prepared. In this sequence, t-BOC-ON
represents t-butoxycarbonyloxyimino-2-phenylacetonitrile and MPL is
as depicted above: ##STR13##
Although t-BOC-ON is employed in the above reaction as the blocking
agent in step (1), other agents can be used as well. Typical
blocking agents include, but are not limited to, benzyl
chloroformate, 9-fluorenylmethyl chloroformate and
2,2,2-trichloromethyl chloroformate.
The blocking of amino acids or other compounds is effected in an
appropriate solvent such as water, ethanol or dioxane, and at
temperatures of from about 0.degree. C. to about 30.degree. C.,
depending upon the nature of the blocking agent.
Activation of the blocked acid in step (2) to form the anhydride
can be accomplished using a compound such as
dicyclohexylcarbodiimide or other compounds such as
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and ethyl
chloroformate. This step is carried out in the presence of an inert
solvent such as chloroform or dichloromethane, and at a temperature
of from about 0.degree. C. to 30.degree. C., depending on the
reagent which is used.
Reaction of the anhydride with MPL in the third step is
accomplished in a solvent mixture, such as pyridine:chloroform 1:1
(V/V), and at a temperature of from about 0.degree. C. to about
30.degree. C.
De-blocking of the amino acid or other compounds introduced into
the MPL molecule can be effected using a de-blocking agent such as
trifluoroacetic acid or hydrogen chloride (gas)in an appropriate
solvent at temperatures within the range of from about -15.degree.
C. to about 0.degree. C.
As previously discussed, the process of this embodiment of the
present invention is particularly useful and provides a unique
method for introducing free amino groups into monophosphoryl lipid
A and related compounds where other functional groups prevent the
direct use of standard coupling agents such as carbodiimides. For
example, MPL contains a phosphate group, a primary hydroxl, and
several secondary hydroxyls. In the presence of carbodiimides or
other condensing agents, side reactions leading to phosphoric
anhydrides, phosphate esters, and other materials occur. The
process of the present invention avoids such side reactions and
accordingly provides a process for preparing the desired
derivatives in relatively pure form.
In a further embodiment, the present invention is directed to
derivatives of monophosphoryl lipid A, wherein a coupling agent has
been attached to the compound of formula (III) to provide a
derivative of the formula: ##STR14## wherein R is as hereinbefore
defined, and A represents a coupling group through which attachment
may be made to the Y group as previously indicated.
Illustrative A groups which can be employed in the present
invention include, but are not limited to, the N-succinimidyl
suberoyl group, the 6-(4'-azido-2'-nitrophenyl) hexanoyl group, the
3-(2-pyridyldithio)propionoyl) group, the m-maleimidobenzoyl group,
and the like.
The reaction of the coupling agents with the compounds of formula
(III) can be effected by known techniques and the particular choice
of conditions will vary according to the particular reactants
employed.
In another embodiment of this invention, derivatives of
monophosphoryl lipid A are provided which are comprised of the
compound of formula (III) above, coupled through the divalent A
groups to biologically active materials. These derivatives
hereinafter also referred to as "conjugates" can be represented by
the formula: ##STR15## wherein A and R are defined above and Y
represents a biologically active material. Such compounds combine
in one molecule the properties of both the biological material and
the monophosphoryl lipid A.
Illustrative biological materials which can be coupled to the MPL
derivative of formula (III) through a coupling agent, include, but
are not limited to: immunopotentiators, such as, Tuftsin, muramyl
dipeptide, cell wall skeleton, trehalose dimycolate, and the like;
cytokines, such as, interleukins, interferons, granulocyte-monocyte
activating factor, tumor necrosis factor, and the like; antigens,
such as, relating to infectious disease; tumor-specific antibodies;
membranes, such as phospholipid membranes, including membranes
composing structures such as liposomes, and carriers, supports,
such as latex beads, dextran, cellulose resins, and the like.
The immunopotentiator, tuftsin, indicated above, and in the
examples, is the compound, L-threonyl-L-lysyl-L-prolyl-L-arginine.
Its pharmacologically acceptable salts and derivatives, and certain
of its optical isomers are useful for stimulating or inhibiting
phagocytosis or pincytosis in mammals. Other therapeutically useful
non-antigenic polypeptides include:
L-thr-L-lys-L-pro-L-arg-L-thr-L-lys-L-pro-L-arg,D-thr-L-lys-L
-pro-D-arg-D-thr-L-lys-L-pro-D-arg,
D-thr-L-lys-L-pro-L-arg-L-thr-L-lys-L-pro-D-arg and
pharmacologically acceptable salts and derivatives thereof.
The coupling of the biologically active component to the MPL
derivative of formula (IX) above can be effected by known reactions
set forth in the literature. In some instances, and depending upon
the functional groups available on the R moiety, it may be possible
to directly link the biologically active component Y directly to R,
and in such instance n in formula (X) above will be zero.
As previously indicated, monophosphoryl lipid A is an
immunostimulant and when coupled to other compounds having
desirable biological properties may impart enhanced biological
activity to such compounds. For example, the effectiveness of MPL
as an adjuvant for antigens may be enhanced when the antigen is
coupled to MPL by employing the derivatives of the present
invention. These conjugates may have utility as more potent
immunopotentiating compounds, better adjuvants for relatively
non-immunogenic antigens, and as tools for immunological research.
The conjugates of the formula indicated above, are accordingly
useful in the detection of various biological components contained
in body fluids, such as blood, urine and also in biological
research. Conjugates, such as those of monophosphoryl lipid A
conjugated to immunopotentiators or cytokines are useful in the
stimulation of specific and non-specific immune response.
Conjugates, having antibodies coupled to MPL are useful in the
stimulation of a specific immune response, in affinity
chromatography, immunoassays, cellular adsorption onto solid
supports and the like. Conjugates having antigens coupled to MPL
are also useful in the stimulations of a specific immune response.
MPL can also be conjugated to carriers for use in biological
research and testing.
The following examples illustrate the best mode presently
contemplated for the preparation of the derivatives of the present
invention.
EXAMPLE 1
Synthesis of N-t-Butoxycarbonyl-6-Aminocaproic Anhydrides
(t-BOC-CAP Anhydride)
N-t-Butoxycarbonyl-6-aminocaproic acid (t-BOC-CAP) was prepared
from t-butoxycarbonyloxyimino-2-phenylacetonitrile (BOC-ON) and
6-aminocaproic acid, following the method of W. J. Paleveda, F. W.
Holly, D. F. Veber, Organic Syntheses 63, 171 (1984). 100 mg
(4.32.times.10.sup.-4 mole) of the resulting t-BOC-CAP was
converted to the anhydride by treatment with 50 mg
(2.42.times.10.sup.-4 mole) of dicyclohexylcarbodiimide in dry
CHCL.sub.3. Following the usual work-up procedures and
recrystallization from diethyl ether/hexane, 76 mg of t-BOC-CAP
anhydride was obtained. (IR-3480, 1821, 1751, 1719 cm.sup.-1 ;
.sup.1 H-NMR - no carboxyl proton).
EXAMPLE 2
Synthesis of (N-t-Butoxycarbonyl-6-Aminocaproyl)-Monophosphoryl
Lipid A (t-BOC-CAP-MPL)
To a 100 ml round bottom flask was added 410 mg (approx.
2.7.times.10.sup.-4 mole) monophosphoryl lipid A dissolved in
chloroform:methanol 4:1 (v/v). The solvent was removed by flash
evaporation, and the flask was left on a lyophilizer overnight to
remove the final traces of solvent and moisture. To the dried
residue was added 171 mg t-BOC-CAP anhydride (3.84.times.10.sup.-4
mole) and 12 mls each of chloroform and pyridine, both of which had
been dried over 4A molecular sieves. The reaction mixture was
stirred under nitrogen. The progress of the reaction was monitored
by thin layer chromatography (TLC) on silica gel 60 TLC plates
(EM), using a solvent system consisting of
chloroform:methanol:water:ammonium hydroxide 50:31:6:2 (v/v).
Developed TLC plates were visualized by spraying with 7%
phosphomolybdate in ethanol followed by charring at 150.degree. C.
The reaction appeared to be complete after the first 24 hours. The
reaction was stopped after 52 hours by adding 30 mls 0.10 M
Na.sub.2 CO.sub.3 (pH 10) and stirring vigorously for 30 minutes.
The mixture was then transferred to a separatory funnel, 12 mls
each of chloroform and methanol were added and the funnel was
shaken. The organic phase was withdrawn, and the aqueous phase was
extracted again with another portion of chloroform:methanol 2:1
(v/v). The organic phases were combined and washed with 1N HCl
until an acidic pH was obtained in the aqueous phase (required 210
mls). The organic phase was washed once with water, then flash
evaporated and taken to final dryness on a lyophilizer. The weight
of the resulting residue was 491 mg.
EXAMPLE 3
Converting t-BOC-CAP-MPL to CAP-MPL
468 mg of t-BOC-CAP-MPL was dried in a 50 ml round bottom flask,
using a lyophilizer to remove the final traces of solvent and
moisture. The flask was then equipped with a magnetic stir bar and
placed in a dry ice/ethylene glycol bath (-15.degree. C.). To the
flask was added 20 mls cold trifluoracetic acid (TFA) which had
previously been dried by vacuum distillation off of P.sub.2 O.sub.5
(distillation flask at -15.degree. C., receiving flask at
-77.degree. C.). The reaction solution was stirred vigorously for
20 minutes, after which time the TFA was removed by distillation.
The final traces of TFA were chased with chloroform, resulting in
450 mg of a glassy reddish-brown residue.
The crude product mixture was fractionated by ion-exchange
chromatography on a 2.5.times.20 cm column of DEAE cellulose in the
acetate form (Indion HA-3). After loading with 447 mg of crude
CAP-MPL, the column was rinsed with 220 mls each
chloroform:methanol 4:1 (v/v) and chloroform:methanol:water 2:3:1
(v/v), and then eluted with a linear salt gradient composed of 900
mls of chloroform:methanol:water 2:3:1 (v/v) against 900 mls of
chloroform:methanol:0.2M ammonium acetate 2:3:1 (v/v). A total of
312 mg of monosubstituted CAP-MPL was obtained in the 4:1 and 2:3:1
foreruns. Another 116 mg of material, corresponding primarily to
unreacted MPL, was recovered during the salt gradient elution.
Further fractionation of CAP-MPL into single components was carried
out on silica gel (BioSil HA), using a linear gradient of 1000 mls
chloroform against 1000 mls chloroform:methanol:water 590:400:10
(v/v). The fractions corresponding to the hexaacyl homolog of
CAP-MPL were pooled and subjected to further purification on 500
micron silica gel H preparative TLC plates (Analtech), using
chloroform:methanol:water:ammonium hydroxide 50:31:6:2 (v/v) as the
developing solvent. Mass spectral analysis of the resulting
purified hexaacyl CAP-MPL revealed a single component with a m/e of
1830, corresponding to the expected mass for hexaacyl MPL combined
with one 6-amino caproyl group.
EXAMPLE 4
Synthesis of (N-Succinimidyl suberoly)-CAP-MPL (SSC-MPL)
Into a 4 ml screw cap vial was placed 14.8 mg hexaacyl CAP-MPL
(8.09.times.10.sup.-6 mole). The vial was charged with 1.0 ml dry
chloroform which had been stored over 4A molecular sieves. To the
chloroform solution of CAP-MPL was added 12.6 mg
di-(N-succinimidyl) suberate (Pierce Chemical Co.;
3.23.times.10.sup.-5 mole), followed by 1.0 ml pyridine (4A sieves)
and a flea bar. The vial was tightly capped, then stirred at room
temperature (24.degree. C.) for several hours until the reaction
was judged complete by TLC. (The product migrates with an R.sub.f
of about 0.49 under the TLC conditions given in Example 2.) At this
point, all solvent was removed by flash evaporation, using an
aspirator to remove the chloroform and a vacuum pump to remove the
pyridine. The temperature was kept below 40.degree. C. at all
times. The resulting residue was transferred to a 30 ml Corex tube
using a minimal amount of chloroform, and product was precipitated
by the addition of 20 ml acetone while vortexing. The precipitate
was pelleted by centrifuging at 12,000.times.g for 20 minutes. The
pellet was suspended in 20 ml acetone and then centrifuged again at
12,000.times.g for 20 minutes. The pellet fraction, after
evaporation of the last traces of solvent, yielded 10.7 mg of
product. (IR-1780 (w), 1810 (w) CM.sup.-1 ; TLC - R.sub.f =0.49 on
silica gel 60, using chloroform:methanol:water:ammonium hydroxide
(50:31:6:2)).
EXAMPLE 5
Coupling of SSC-MPL to Tuftsin (Thr-Lys-Pro-Arq)
10.1 mg partially purified SSC-MPL was dried into a 5 ml round
bottom flask, resulting in a thin, clear film. 16.7 mg tuftsin
(Sigma; 3.34.times.10.sup.-5 mole) was dissolved in 1.37 ml 100 cm
HEPES buffer at pH 7.85, which had been prepared by titrating the
free acid form of HEPES (Sigma) with triethylamine. The total
quantity of the aqueous tuftsin solution was added to the flask,
along with a 10.times.3 mm magnetic stir bar, and the solution was
vigorously stirred overnight. The solution was occasionally
submitted to brief (ca. 10 seconds) sonication in an ultrasonic
bath to facilitate disruption of the SSC-MPL film. After 24 hours,
the solution was transferred to a dialysis bag (cutoff--6000-8000
MW) and dialyzed exhaustively against distilled water. The
resulting dialysate was lyophilized, ultimately yielding 12.3 mg of
a white powder. (TLC - R.sub.f =0.12, 0.21 (major) and 0.45 (minor)
on silica gel 60, developed with chloroform:methanol:water 65:25:4.
SSC-MPL migrates at R.sub.f =0.53 in this TLC system.)
The crude product mixture was fractionated by preparative TLC on
500 micron silica gel H prep plates (Analtech), using the 50:31:6:2
solvent system to develop the plates. Bands were visualized by
backlighting, and the products were recovered by standard
techniques. Two relatively pure product bands were recovered,
corresponding to R.sub.f =0.12 (2.3 mg) and R.sub.f =0.21 (4.5 mg)
in the 65:25:4 TLC system.
EXAMPLE 6
Synthesis of Succinoyl-MPL
To a 2.0 ml screw-cap vial was added a chloroform:methanol 4:1
(v/v) solution containing 29 mg (1.68.times.10.sup.-5 mole)
hexaacyl MPL. All solvent was evaporated, the final traces being
removed on a lyophilizer. 3.4 mg (3.36.times.10.sup.-5 mole)
succinic anhydride was weighed into the vial and a flea bar was
added, followed by 0.2 ml each of chloroform and pyridine which had
been dried by storing over 4A molecular sieves. The solution was
stirred for 5 hours, then transferred to a 15 ml Corex centrifuge
tube and quenched by stirring with 1.0 ml 0.1 M Na.sub.2 CO.sub.3
(pH 10.0) for minutes. 2 mls chloroform and 1 ml methanol were
added to the quenched reaction, and it was centrifuged at 3,000 rpm
for 10 minutes to separate the phases. The organic layer was then
washed 2 times with 1N HCl, one time with water, and evaporated
under a stream of nitrogen to yield 27.4 mg of a glassy residue. As
estimated by silica gel TLC (see Example 2, above), this material
consisted of 50% mono-succinyl-MPL, 20% di-succinyl-MPL, and 30%
unreacted MPL.
Although the invention has been illustrated by the preceeding
examples it is not to be construed as being limited to the
materials employed therein; but rather, the invention encompasses
the generic area as hereinfore disclosed. Various modifications and
embodiments can be made without departing from the spirit or scope
thereof.
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